The present invention relates to tetrazolium chromagens and their use in histological staining and in enzyme amplified assays.
Tetrazolium halides have been widely used as stains, especially in histology. Such compounds have a cationic tetrazolium ring, three phenyl substituents at the 2, 3 and 5 positions on the ring and a halide anion (often chloride or bromide). The chromagen is reduced to a formazan, which is strongly colored and is insoluble in water. For the derivatives with one, two or three 4-nitrophenyl substituents (and correspondingly two, one or no phenyl substituents), the ease of reduction to formazan increases with increasing numbers of nitrophenyl substituents. Literature references indicate that a 4-nitrophenyl substituent attached to the 2-position or 3-position of the tetrazolium ring increases the reducibility to a greater extent than does the same group linked to the 5-position of the tetrazolium ring. One reference suggests that, in fact, a 4-nitrophenyl group attached at the 5-position of the tetrazolium ring has the opposite effect compared to that same group attached to the 2-position or 3-position. See F.P. Altman, Prog.
Histochem. Cytochem., vol. 9, pp. 1-56 (1976); F.P. Altman, Histochem. J., vol. 8, pp. 471-85 (1976); E. Seidler, Z. Med. Labortechnik, vol. 9, pp. 241-51 (1968); Histo-Und Zytochemie Dehydrierender Enzyme; Grundlagen Und Problematik (F. Wohlrab et al, eds., (1979), pp. 9-159 and 257-92; and E. Seidler, Histochem.
J., vol. 12, pp. 619-30 (1980).
A widely used tetrazolium halide is 2-[4-iodophenyl)-3-[4-nitrophenyl)-5-phenyltetrazolium chloride, known as INT. INT has the advantages of high stability in aqueous solutions. Once reduced to the corresponding formazan, a strong reddish-brown color is formed. The formazan is stable to reoxidation or dissolution and is therefore a very satisfactory histological stain.
INT has also been used as chromagen to develop an enzyme localized on tissue (whether localized by antigenic determinants or by hybridization on nucleic acids). For example, the localized enzyme alkaline phosphatase (AP) hydrolizes 5-bromo-4-chloro-3-indolylphosphate (known as BCIP) to a reactive intermediate, which reduces INT to the formazan. Thus, when tissue with localized AP is contacted with BCIP and INT in aqueous media (the chromagen), reaction occurs leading to the red formazan being deposited on the tissue in the vicinity of the localized enzyme. See J. McGadey, J. Med. Lab. Technol. vol. 24, pp. 126-28 (1967); J. McGadey, Histochemie, vol. 23, pp. 180-84 (1970).
While INT is a satisfactory chromagen for both histological staining and such enzymatic assays, its reduction to formazan is somewhat difficult. In some situations, that results in some spreading of the reaction product away from the desired site (i.e., the site of immobilized AP enzyme).